Argininosuccinate lyase: purification and characterization from human liver.
Argininosuccinate lyase has been purified to near homogeneity and partially characterized from extracts of human liver. The purified enzyme had a specific activity of 10.3 mumol min-1 mg-1 in the forward, argininosuccinate cleaving, reaction and 8.0 mumol min-1 mg-1 in the reverse reaction. On the basis of electrophoretic mobility in sodium dodecyl sulfate containing polyacrylamide gels, the protein had a minimum molecular weight of 49 000. Sedimentation equilibrium centrifugation revealed a molecular weight of 187 000. On the basis of these data, the enzyme appears to be a tetramer composed of subunits of identical molecular weight. The Km values were about 0.20 mM for argininosuccinate, 5.3 mM for fumarte, and 3.0 mM for arginine. The enzyme exhibited normal Michaelis-Menten kinetics, and guanosine 5'-triphosphate (GTP) had no affect on the activity of the enzyme. With the exception of its kinetic properties, the enzyme is very similar to the beef liver enzyme. Antibodies were prepared in rabbits and were specific for the human protein, reacting only slightly with the beef liver enzyme and not at all with the rat liver enzyme. The antibodies reacted identically with purified enzyme and enzyme in extracts of human skin fibroblasts. Immunoadsorption of crude human liver extracts followed by analysis of the immunoprecipitates by sodium dodecyl sulfate gel electrophoresis showed only one protein band which corresponded in mobility to purified argininosuccinate lyase, demonstrating that the antibodies were specific for argininosuccinate lyase.
D000818:Animals; D001123:Argininosuccinate Lyase / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007118:Immunoassay; D005779:Immunodiffusion; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008190:Lyases / Q000302:isolation & purification*; D046911:Macromolecular Substances; D008970:Molecular Weight; D012867:Skin / Q000201:enzymology
D046911:Macromolecular Substances; D008190:Lyases; D001123:Argininosuccinate Lyase